NT4-ADNF-9融合基因原核表达载体的构建

目的　构建神经营养素 (NT4 )与活性依赖性神经营养因子 9(ADNF 9)融合基因的原核表达载体pBV2 2 0 /NT4 ADNF 9,为进一步对神经系统退行性疾病基因治疗的研究奠定基础。方法　采用非对称互补引物 /模板法 ,制备两端含有酶切位点的ADNF 9的cDNA ,将其连接到NT的信号肽和前导序列的 3′端 ,组成融合基因NT4 ADNF 9,再将该融合基因亚克隆于原核表达载体pBV2 2 0 ,构建为pBV2 2 0 /NT4 ADNF 9。结果　经DNA测序 ,限制性内切酶酶切等方法证实已成功地将ADNF 9重组到NT4信号肽和前导序列的 3′端 ,并将融合基因亚克隆于pBV2 2 0内。结论　成功构建了NT4与ADNF 9融合基因的原核表达载体pBV2 2 0 /NT4 ADNF 9。

Construction of expression vector for NT4-ADNF-9 fusion gene

Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay a foundation for the further study on genetic therapy of neurodegenerative disorders. Methods By means of asymmetrical primer/template, double stranded cDNA of ADNF-9 was gained, which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4(NT4), the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220, then called pBV220/NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcloned into pBV220 successfully. Conclusion Prokaryotec expression vector pBV220/NT4-ADNF-9 can be constructed successfully.